**2.2. The Barrier: Second and third lines of defence**

The primary role of skin is to provide a physical barrier against the environment and this presents the first line of defence. However, crucial immune components form integral elements of the skin, providing the subsequent layers of defence with increasing specificity. Potential microbial invaders will be confronted by the physical, innate (non-specific), chemical and granulocyte barrier (adaptive or specific immune system) that evolved over time to eradicate these invaders, digest invading cells into smaller antigens, and finally to programme lymphocytes in order to provide long term protection against the particular microbe [13].

A microbe that manages to penetrate the subcutaneous layers of the skin will be met by the second line of defence; the innate or non-specific immune system. This defence mechanism involves the production pro-inflammatory substances, such as chemokines by the lipokeratinocytes, resulting in the migration of immune cells such as leucocytes to the site of infection. Leucocytes include among others, the phagocytes (macrophages, neutrophils and dendritic cells). Upon detection of the foreign proteins originating from potential bacterial, fungal or parasitic invaders, Langerhans cells will phagocytise these antigens and migrate to adjacent lymph nodes. In the lymph nodes, these cells will develop into mature dendritic cells which will process the antigen (into smaller fragments), to activate the adaptive immune system (lymphocytes) [14]. On top of this chemical defence mechanism, nonspecific antimicrobial substances such as lysozyme and the peptide β-defensin were found in cetacean integument [15]. It was found that lysozyme occurs between the layers of the stratum corneum, within cells of the stratum spinosum, dermis and endothelial cells of the dermal blood vessels. Also, β-defensin was found to be concentrated in the upper five or six layers of the stratum corneum, as well as within the cells of the upper stratum spinosum.

Cutaneous Lesions in Cetaceans: An Indicator of Ecosystem Status? 127

larger size of hyphae prevent them from being ingested. Over time, pathogenic fungi evolved and developed different strategies to survive and even disseminate in mammalian tissue. The neutrophils, macrophages and monocytes are the main antifungal effector cells in the defence strategy, employing shared mechanisms [17]. Firstly, macrophages present at the site of attempted infection, will make an effort to damage or kill the fungus. The second line of defence includes effector cells, the neutrophils and monocytes, which are summoned to the infection site by inflammatory signals from cytokines, chemokines and complement components. These effector cells damage or kill the fungal invader using strategies such as

There are four main groups of fungal infections [16]. The first, superficial mycoses, does not provoke any immune response from the host since the fungus would only grow on compounds associated with the skin. The remaining three categories provoke immune responses and include cutaneous, subcutaneous and deep mycoses. In cases of local trauma, subcutaneous mycoses may develop with the subcutis as the primary site of infection. In such cases, leucocytes and eosinophils will respond, leading to the formation of cysts or granulomas. Deep mycoses usually occurs in immuno-suppressed mammals, with entry through the lungs, paranasal sinuses, digestive system, or mucous membranes. Total fungal

A limited number of organisms have the ability to degrade and utilize keratin, the key structural component of mammalian skin [18]. These include a few insect species, as well as a number of bacteria and fungi. Higher vertebrates are also not known to digest keratin [11]. Microbes degrade keratin by the secretion of extracellular proteolytic enzymes, known as keratinases; members of the serine proteinase group of enzymes [19, 20]. These enzymes are robust with a wide temperature and pH activity range, and have the ability to hydrolyze both natural and denatured keratin [21]. Keratinases from the fungus *Microsporum gypseum* were found to cleave the disulfide bridges in the keratin (sulfitolysis), which were followed by a further attack on the keratin structure by extracellular proteases [18]. Tsuboi and coworkers [22] found a keratinolytic proteinase in another fungus, i.e. *Trichophyton mentagrophytes,* with an optimal pH of 4.5 for keratin and 3.9 for haemoglobin. They showed that this fungus could potentially invade healthy skin (with a weakly acidic pH), by breaking down the keratin and thereby making it possible for the organism to invade the stratum corneum. This ability to degrade keratin, and other molecules associated with skin,

Keratinophilic (keratin loving) fungi (Figure 2) represent the largest group of organisms with the ability to degrade and utilize keratin as a source of carbon and nitrogen [11, 16]. These fungi commonly occur in soil and sewage sludge, which contain high concentrations of keratin remnants with specific physiochemical properties and associated microbial populations [23]. Releasing sewage sludge into the environment, or using it for fertilizing purposes, can therefore lead to spreading of potentially pathogenic fungi into new

producing reactive oxygen intermediates and antimicrobial peptides [17].

dissemination is usually a sign of severe immune failure.

can be regarded as a putative microbial virulence factor [20].

**3. Microbes – The enemy?** 

In addition to the above-mentioned non-specific defence mechanisms, intra-epidermal lymphocytes indicate the presence of the adaptive immune system in the epidermis of mammals. These specialized T cells have powerful cytolytic and immuno-regulatory effects on antigens and will confine antigens that overcame the first and second lines of skin defence [14]. Cells associated with this specific or adaptive immune response were also detected in cetacean skin studies [13]. Zabke and Romano [13] reported that their study on dolphin skin revealed the presence of MHC II (+) antigen cells, predominantly situated in the dermal papillae, along the epidermal-dermal border. These cells were found to have a dendritic-like morphology and form patterns, similar to those of Langerhans cells. The latter are known primary antigen presenting cells in the integument of terrestrial mammals, and thus the authors concluded that they were most likely Langerhans cells and not macrophages or dendritic cells. The latter two types were found deeper into the dermis. Zabke and Romano [13] further suggested that pathogen invasion resulting from a wound may lead to an inflammatory response, causing immune cells (neutrophils, macrophages and/or lymphocytes) to migrate from the dermal papillae to the site of infection. However, the authors indicated that inflammation is usually absent in these animals, because this barrier is normally sufficient against small injuries sustained via interactions with other dolphins. The authors further noted that wound healing in dolphins is not accompanied by scab formation. In these mammals hydrophobic changes within in the stratum spinosum, causes rapid sloughing and replacement with cells of the stratum germinativum undergoing mitosis.

From the above it is evident that cetacean skin is an effective physicochemical barrier. To overcome such a challenging obstacle would require a failure of the barrier itself, or creative strategies and unique properties in the prospective invader.

*Immune response to cutaneous fungal infections.* The type of fungal invader, whether a unicellular yeast or hyphal fungus, as well as anatomical site of infection, will determine the immune response of the host [16,17]. Yeast cells are usually phagocytosed, whereas the larger size of hyphae prevent them from being ingested. Over time, pathogenic fungi evolved and developed different strategies to survive and even disseminate in mammalian tissue. The neutrophils, macrophages and monocytes are the main antifungal effector cells in the defence strategy, employing shared mechanisms [17]. Firstly, macrophages present at the site of attempted infection, will make an effort to damage or kill the fungus. The second line of defence includes effector cells, the neutrophils and monocytes, which are summoned to the infection site by inflammatory signals from cytokines, chemokines and complement components. These effector cells damage or kill the fungal invader using strategies such as producing reactive oxygen intermediates and antimicrobial peptides [17].

There are four main groups of fungal infections [16]. The first, superficial mycoses, does not provoke any immune response from the host since the fungus would only grow on compounds associated with the skin. The remaining three categories provoke immune responses and include cutaneous, subcutaneous and deep mycoses. In cases of local trauma, subcutaneous mycoses may develop with the subcutis as the primary site of infection. In such cases, leucocytes and eosinophils will respond, leading to the formation of cysts or granulomas. Deep mycoses usually occurs in immuno-suppressed mammals, with entry through the lungs, paranasal sinuses, digestive system, or mucous membranes. Total fungal dissemination is usually a sign of severe immune failure.
